Human exposure to Pneumocystis carinii is common but, in the absence of acquired or genetic dysfunction of either cellular or humoral immunity, exposure rarely leads to illness. Although alveolar macrophages can degrade P. carinii, macrophage receptors involved in P. carinii recognition have not been clearly defined. Characterization of a predominant surface glycoprotein of the high mannose type led us to investigate the role of the macrophage mannose receptor in this process. We report here that binding and uptake of cultured rat P. carinii by human and rat alveolar macrophages is reduced by 90% in the presence of competitive inhibitors of mannose receptor activity and by adherence of alveolar macrophages to mannan-coated surfaces. Further, only those COS cells transfected with the human macrophage mannose receptor complementary DNA that express surface mannose receptors bind and ingest P. carinii. These studies establish that the macrophage mannose receptor is sufficient for uptake of P. carinii and emphasize the role of the alveolar macrophage in first-line host defence against P. carinii.

The macrophage mannose receptor, a pattern recognition molecule and component of innate immunity, mediates binding and phagocytosis of Pneumocystis carinii and likely represents an important clearance mechanism in the lungs of immunocompetent hosts. The purpose of this study was to examine the ability of alveolar macrophages from HIVinfected individuals to bind and phagocytose P .

Although originally classified as a protozoan, Pneumocystis carinii is now considered to have fungal characteristics. Drugs typically used for the treatment of fungal infections target ergosterol. Because P. carinii is an important pathogen in AIDS and other immunocompromised patients, knowledge of the sterol content of this organism may be useful as a basis for developing new treatment strategies or for improving diagnosis. P. carinii organisms were harvested from infected rat lungs and were purified by filtration. Control preparations from uninfected animals were identically prepared. Lipids were extracted from the organisms and control preparations and were separated into neutral lipid, glycolipid, and phospholipid fractions by silicic acid chromatography. The neutral lipid fraction was further treated by alkaline hydrolysis and was analyzed by reversed-phase high-pressure liquid chromatography (HPLC), gas chromatography (GC), and GC-mass spectrometry (GC-MS). As shown by HPLC, the neutral lipid fraction from infected rats contained a minimum of six peaks, while in control preparations a single peak with a retention time identical to that of cholesterol was observed. The predominant sterol in these preparations was positively identified by GC-MS as cholesterol and constituted 80 to 90% of the total. The remaining peaks had relative retention times similar to those of phytosterols by both HPLC and GC, and the similarity of these sterols to those derived from plants and fungi was confirmed by MS. Ergosterol, however, was not present. These results provide further evidence for a close phylogenetic relationship between P. carinii and fungi and suggest that these sterols could be used as targets for drug development and for improving diagnosis.Pneumocystis carinii is one of the leading causes of opportunistic infection in AIDS patients (30). Despite the importance of this microorganism as an opportunistic pathogen, much basic information about the parasite is lacking. While originally classified phylogenetically as a protozoan, analyses of P. carinii DNA and mitochondrial and rRNA sequences have generally shown a higher degree of homology to the mitochondrial and rRNA sequences of fungi (yeasts) than to those of protozoa (9,13,27,31,34,35). The exact relationship remains to be determined. Analyses of 5S RNA have suggested a somewhat close phylogenetic relationship to members of the Rhizopoda-Myxomycota-Zygomycota group of "protista fungi." Other studies have suggested similarities to Saccharomyces, Candida, and Neurospora spp. and "red yeast" fungi. In general, the arguments in favor of classifying P. carinii as a protozoan are the amoeboid appearance of the trophic form, susceptibility to antiprotozoal drugs such as pentamidine isethionate or trimethoprim-sulfamethoxazole, insusceptibility to antifungal drugs such as amphotericin B, and the lack of ergosterol in Pneumocystis cell membranes. Arguments in favor of including P. carinii among the fungi include similarities of the sporogenous state to ascospore format...

Respiratory infection with Pneumocystis carinii (PC) is the most frequent serious opportunistic infection in the clinical setting of acquired immunodeficiency syndrome (AIDS). The factors responsible for the predisposition of human immunodeficiency virus (HIV)-infected patients for PC infection are not fully understood. We postulated that changes in the alveolar lining material (ALM) could play a role in the pathogenesis of PC infection in AIDS. We have compared constituents of ALM in bronchoalveolar lavage fluid from normal, nonsmoking volunteers with that of HIV-infected patients with pneumonia. Using an ELISA, we found that surfactant protein A (SP-A) was markedly elevated in the pneumonia patients. Mean SP-A values for the normal nonsmoking individuals (n = 21) were 1.50 +/- 0.25 micrograms/ml (mean +/- SEM). SP-A levels in the HIV-infected patients (n = 22) were significantly elevated (p less than 0.01) with a mean of 5.23 +/- 0.54 micrograms/ml. This increase was greatest in the patients with more clinically severe pneumonia. The increase in SP-A did not appear to be pathogen-specific as it was also observed in cases of non-PC pneumonia. We also found that total protein levels were nearly five times higher in the HIV-infected pneumonia patients. These studies indicate that the protein component of the ALM is markedly different from normal in cases of HIV-associated PC and non-PC infection. Further investigation is needed to determine the mechanism of these alterations and their role, if any, in AIDS-related pneumonia.

Published and unpublished data on the cultivation of P. carinii were reviewed by a panel of investigators convened by the National Institutes of Health. Although several cell culture systems allow propagation of P. carinii for a limited time with modest rates of replication, these have not proved adequate for isolation of P. carinii in sufficient quantity to explore important basic biological investigation. Attempts at cell-free culture have yielded only transient proliferation. Because much of the unsuccessful work on cultivation of the organism has been unpublished, the panel agreed that these data may be useful to other investigators in designing experimental strategies for cultivation. Therefore, the purpose of this report is to make available this information to researchers, lest others unknowingly repeat unsuccessful methods. It is hoped that by documenting the history and the complexities of Pneumocystis culture, renewed interest and efforts will be directed toward this fundamental scientific challenge.

Intracellular dissolution of inhaled particles is an important pathway of clearance of potentially toxic materials. To study this process, monolayers of human and canine alveolar macrophages (AM) were maintained alive and functional in vitro for more than 2 wk. Complete phagocytosis of moderately soluble, monodisperse 57Co3O4 test particles of four different sizes was obtained by optimizing the cell density of the monolayer and the particle-to-cell ratio. The fraction of the initial particle mass that was soluble increased over time when the particles were ingested by AM but remained constant when in culture medium alone. Smaller particle sizes had a faster characteristic intracellular dissolution rate constant than did larger particles. The dissolution rates differed between AM obtained from two human volunteers as compared to those obtained from six mongrel dogs. These in vitro dissolution rates were very similar to in vivo translocation rates previously obtained from human and canine lung clearance studies after inhalation of the same or similar monodisperse, homogeneous 57Co3O4 test particles. We believe an important clearance mechanism for inhaled aerosol particles deposited in the lungs can be simulated in vitro in a cell culture system.

The alveolar macrophage (AM) oxidative burst response is an important component of microbicidal effector cell function against a variety of potential pathogens in the lungs, although the role against Pneumocystis carinii has not been fully investigated. The goals of this study were to characterize the P. carinii-mediated oxidative burst of AMs from healthy individuals, and to examine the oxidative burst of AMs from human immunodeficiency virus (HIV)-infected persons. For healthy individuals, the AM oxidative burst (measured as hydrogen peroxide [H(2)O(2)] production) increased in a time- and concentration-dependent manner in response to P. carinii or to the major surface glycoprotein of P. carinii, gp-A (0.01 to 10 microg/ml), required physical contact of P. carinii with AMs, and was not dependent on organism viability. Enzymatic removal of the surface-associated molecules of P. carinii reduced the oxidative burst to 43% of control (P = 0.01). Blocking the AM mannose receptor reduced the P. carinii-mediated oxidative burst response to 37% of control (P = 0.01). Compared with AMs from healthy individuals, P. carinii-mediated H(2)O(2) production was significantly reduced in AMs from asymptomatic HIV-positive (HIV+) persons with CD4+ counts < 200 cells/mm(3) (249+/-43 relative fluorescence units [RFU] versus 130+/-44 RFU; mean +/- standard error of the mean, P = 0.038) and HIV+ persons with active P. carinii pneumonia (78+/-40 RFU; P = 0.014), but preserved for HIV+ persons with CD4+ counts > 200 cells/mm(3). Importantly, H2O2 production in response to phorbol myristate acetate or serum-opsonized zymosan particles was preserved in all groups studied. Thus, AM oxidative burst, mediated in part via P. carinii gp-A and AM mannose receptor may represent an important host response to P. carinii. A specific impairment of P. carinii-mediated AM oxidative burst in persons with advanced HIV infection may contribute to the pathogenesis of P. carinii pneumonia.

Threshold estimates for multiple-interval forced-choice staircase procedures were studied using computer simulations. A sigmoidal psychometric function shape governed the hypothetical subject's responses in the simulations. Parameters varied included the number of trials, the step size for stimulus level change, and decision rules that targeted 70.7% and 79.4% correct performance. Each threshold estimate was calculated by averaging the stimulus levels at which a reversal a stimulus level direction occurred. The results of the simulations suggest that, as the number of alternatives is increased from 2 to 4, the variability of repeated threshold estimates decreases or remains constant, and the accuracy of the estimator, in most cases, improves. A subset of the simulations was compared with data obtained in a detection-in-noise task. The behavioral data were consistent with the simulation results. Two major conclusions were reached. First, 3- and 4-interval forced-choice (IFC) procedures are more efficient than a 2IFC procedure with a decision rule that targets 70.7% correct performance even when the additional time required to complete 3- and 4IFC trials is considered. Second, the accuracy of 2IFC procedures can be improved by fitting the trial history of a staircase run using probit analysis.